Live bovine adenovirus vaccines, preparation thereof and method of vaccination using them

ABSTRACT

The invention relates to virus vaccines containing temperaturesensitive mutant strains of bovine Adenovirus, the cut-off temperature of said mutant strain being comprised between 39* and 40* C. The invention also relates to the induction of said temperaturesensitive mutant strains and to a vaccination method by intranasal administration of the obtained vaccines.

United States Patent [191 Zygraich et al.

[ Dec. 16, 1975 LIVE BOVINE ADENOVIRUS VACCINES,

PREPARATION THEREOF AND METHOD OF VACCINATION USING THEM [75] Inventors:Nathan Zygraich, Brussels; Constant Huygelen, Huldenberg, both ofBelgium [73] Assignee: Recherche et Industrie Therapeutiques (R.I.T.),Belgium [22] Filed: Jan. 31, 1974 [21] Appl. No.: 438,503

Related US. Application Data [63] Continuation-impart of Ser. No.360,219, May 14,

1973, abandoned.

[52] US. Cl. 424/89; 195/1.1 [51] Int. Cl. A61K 39/12 [58] Field ofSearch 424/89; l95/l.1

[56] References Cited UNITED STATES PATENTS 9/1962 Gierer et al 424/89OTHER PUBLICATIONS Mattson, Am. J. Vet. Res. 34(5): 623-629, May 1973,

Naturally Occurring Infection of Calves with a B0- vine Adenovirus.

Bartha, Vet. Bull. 38, No. 147 (1968), Immunization Experiments onCalves with Type 45 Bovine Adenovirus.

Williams et al., Chem. Abst. 76, No. 149458q (1972),Temperature-restricted Mutants of Human Adenovirus Type 5, Strategy ofthe Viral Genome, Ciba Fndn. Sympos. (1971), pp. 275-290.

Primary Examiner-Shep K. Rose Attorney, Agent, or Firm-Alan D. Lourie;William H. Edgerton 57] ABSTRACT 6 Claims, No Drawings LIVE BOVINEADENOVIRUS VACCINES, PREPARATION THEREOF AND METHOD OF VACCINATION USINGTHEM The present invention which is a continuation-impart of ourcopending application Ser. No. 360,219 filed on May. 14, 1973 nowabandoned relates to the preparation of temperature-sensitive (ts)mutant strains of bovine Adenovirus strains which are valuable for thepreparation of vaccines, to the live virus vaccines containing saidtemperaturesensitive (ts) mutant strains and to a vaccination methodused said live virus vaccine.

Essentially, the substantial in vitro replication of atemperature-sensitive (ts) mutant strain is limited to temperaturesinferior to its cut-off temperature. Although results obtained in vitrocannot necessarily be extrapolated to what occurs in vivo, some reportssuggest that, in vivo, ts mutants behave differently from wild viruses(B. R. Murphy, E. G. Chalhud, S. R. Nusinoffand R. M. Chanock, J. oflnf. Dis. 126, no. 2, 170-8, 1972).

This phenomenon has been applied to the development of some live virusvaccines against respiratory diseases. At the best ts conditions, a tsmutant with a cut-off temperature in the range of the normal bodytemperature should be able to multiply in the mucosae of the upperrespiratory tract (where the temperature is several degrees C lower thanthat of the lower respiratory tract and body) while it replication wouldbe partially or completely inhibited in the lower respiratory tract andin the body. Studies in laboratory animals and in man have shown that,at least for some respiratory viruses, this theoretical approach isconfirmed by the experimental findings (forexample, N. Zygraich, M.Lobmann and C. Huygelen, J.Hyg.Camb. 70, 22934, 1972). Nevertheless, andmore particularly because of the high specificity existing in thisfield, iit was not obvious that the same principle could be applied toother viruses, more particularly bovine Adenoviruses e.g. Adeno 3 virusin order to provide mutant strains valuable for the production ofimmunogenic and non-pathogenic live bovine Adenovirus vaccines.

This procedure circumvents the obstacles which up to now were facing thepreparation of live bovine Adenovirus vaccines, i.e. the pathogenicityand side effect generating of the bovine Adenoviruses on the respiratorytract and on the gastro-intestinal tract or on the eye respectively.

We have now surprisingly found that when inducing in very specificconditions temperature-sensitive (ts) mutant strains of bovineAdenoviruses e.g. bovine Adeno 3 virus bovine Adenoviruses mutantstrains having a cut-off temperature comprised between 39 and 40 C areisolated which are particularly suitable for vaccine use or production,owing to the fact that they are simultaneously genetically stable,immunogenic, non-pathogenic and that their administration to susceptibleanimals involves no detectable side effect.

A further advantage of the vaccines of this invention resides in theiradministration route. The vaccines of this invention indeed areadministered intranasally; their constituting bovine Adenovirus strainsmultiply only locally in the upper respiratory tract of the animals,without any detectable virus multiplication at the warmer temperaturesof the organism, i.e. in the internal organs of the animals.

For preparing the non-pathogenic bovine Adenovirus strains useful forvaccine production according to this invention, .either a bovineAdenoviruse.g. bovine Adeno 3 virus-strain directly isolated from aclinical case or a bovine Adenovirus strain obtained at the end ofserial passages in tissue culture such as for instance primary bovinekidney (PBK) cell culture (such as WBR 1 strain J. H. Darbyshire et al.Nature 208, 3078, 1965- or primary foetal bovine kidney (PFBK) cellculture may be used as starting material.

The present invention does comprise a process for preparing anon-pathogenic bovine Adenovirus e.g. bovine Adeno 3 virus strain from apathogenic bovine Adenovirus-strain consisting in inducing therefrom andisolating a temperature-sensitive mutant strain of bovine Adenovirushaving a cut-off temperature comprised between 39 and 40 C.

According to the invention, the induction of temperature-sensitivemutant strains is performed by bringing bovine Adenovirus e.g. Adeno 3virus strain into contact with a buffered aqueous solution of nitrousacid at room temperature and at a pH comprised be tween 4.2 and 5. Thebuffered aqueous solution of nitrous acid is preferably nitrous acid inacetic buffer, the concentration of nitrous acid and acetate ion in thereaction medium being N and N/4 respectively, the contact being thenmaintained for 2 to 5 (i 1) minutes at pH 4.2 (:01) up to 15 to 25minutes (i 1) at pH 5 (i 0.1); for instance, the contact is maintainedfor 15 minutes (i 1) at pH 4.6 (i 0.1).

It is obvious that pH and temperature conditions are interrelated butthe limits of the above defined conditions are such that an initialvirus population of about 10 is reduced to about 10 after the mutagenictreatment and allow induction of temperature-sensitive mu tant strainshaving a cut-off temperature comprised between 39 and 40 C.

The so-obtained temperature-sensitive (ts) mutant strains are thenisolated by at least one passage in any tissue culture known to the artfor accepting growth of said bovine Adenovirus.

For instance, isolation is possibly performed by at least one clonepassage in primary foetal bovine kidney (PFBK) cell culture, said clonepassage being conducted at 30 C (i 1 C) for a periodof time compris ingbetween 10 and 20 days, preferably for 14 days (i l).

The isolated strains are then optionally further pas saged in any tissueculture known to the art for accepting growth of said bovine Adenovirussuch as kidney cell cultures of bovine origin, and more particularlyprimary foetal bovine kidney cell cultures e.g. two or three times inprimary foetal bovine kidney (PFBK) cell cultures in order to get asubstantial amount of said temperature-sensitive mutant strains.

An example of starting material is a pathogenic strain of bovine Adeno 3virus obtained from the WBR 1 strain after seven serial passages onprimary foetal bovine kidney cell cultures and herein referred to as WBR1/7 strain. A genetically stable non-pathogenic strain of bovine Adeno 3virus obtained according to the process of the present invention hasbeen named Eunice strain in applicants collection where WBR H7 andEunice strains samples can be obtained upon written request.

The temperature-sensitive bovine Adenovirus strains obtained by thisinvention e.g. the Eunlfi strainshow no substantial loss ofimmunogerilEli tion. Consequently, the present invention relates to livebovine Adenovirus vaccines e.g. Adeno 3 virus vaccine containing atleast one bovine Adenovirus strain obtained by inducing and isolating bythe herein described process a temperature-sensitive bovine Adenovirusmutant strain and to the process of preparing said vaccine therefrom.

According to this embodiment, the invention relates to a process forpreparing a live bovine Adenovirus vaccine e.g. an Adeno 3 virus vaccinecomprising incubating a temperature-sensitive bovine Adenovirus having acut-off temperature comprised between 39 and 40 C in a tissue cultureknown to the art for accepting growth of bovine Adenovirus e.g. primaryfoetal bovine kidney (PFBK) cell culture at a temperature not exceeding37 C (t 1C) and preferably ranging 35 C (i 1C) and for a period of timesufficient to permit growth of a large amount of said virus, andharvesting the resulting virus material More particularly, the saidtemperature-sensitive and non-pathogenic bovine Adenovirus e.g. bovineAdeno 3 virusis obtained as indicated above.

For the purpose of industrial and multiple batches production of thevaccine, the above production step of a large amount of said virus mayobviously comprise more than one passage, the intermediate one or onesbeing then provide for the purpose of virus seed production to be usedfor preparing vaccine batches.

The so-obtained live bovine Adenovirus vaccines are administeredtopically in the nasopharynx at an effective dosage unit e.g. 2.10 TCID(tissue culture infection dose 50 of Adeno 3 virus.

For vaccinal use, the virus is preferably kept in freeze-dried form andthe vaccine is extemporaneously reconstituted by addition of eitherwater or any other pharmaceutical diluent or composition known to theart for the preparation of nasal preparations such as coarse spray.

The invention thus also relates (1) to live bovine Adenovirus e.g.bovine Adeno 3 virus vaccines containing as active ingredient atemperature-sensitive bovine Adenovirus having a cut-off temperaturecomprised between 39 and 40 C e.g. bovine Adeno 3 virus Eunice strainsaid vaccines being more particularly obtained according to thehereabove described process, (2) to polyvalent bovine respiratory livevirus vaccines adrninistrable by intranasal route containing as activeingredients said temperature-sensitive bovine Adenovirus vaccine and atleast one other bovine respiratory live virus e.g. infectious bovinerhinotracheitis virus and/or bovine parainfluenza 3 virus vaccineadrninistrable by intranasal route and (3) a vaccination methodconsisting in administering to a susceptible organism an effective doseof said temperature-sensitive mutant strain of bovine Adenovirus.

The following examples illustrate the present invention; they should notbe construed as limiting its scope.

EXAMPLE 1 Bovine Adeno 3 virus WBR 1/7 strain is suspended in tissueculture Eagles minimal maintenance medium (MEM) to yield a virussuspension containing TCID (tissue culture infective dose 50 One ml. ofthis virus suspension is mixed with 0.5 ml. of a 4 M sodium nitriteaqueous solution in 0.5 ml. of molar acetic acid/sodium acetate buffer(prepared by mixing of glacial acetic acid (6 g) up to 100 ml. withdistilled water and 3 volumes of a solution of sodium acetate (13.6 g.)in 100 ml. of distilled water, both solutions being sterilized for 30minutes at 121 C), the final pH being 4.6

The mixture is allowed to react for 15 minutes at room temperature andthe reaction is then stopped by dropwise addition of normal sodiumhydroxide with stirring up to reaching pH 7.5 (i 0.5). The pH adjustmentis followed by changing of color of the phenol red indicator present inthe virus suspension.

The medium is immediately dialyzed for 5 hours at 4 C (i 1) againstphosphate buffer saline (consisting of NaCl (8 g.); KCl (0.2 g.); Na HPO(1.15 g.); KHJO, (0.2 g.) in distilled water (up to 800 ml.) mixed witha solution of MgCl .6H O in 100 ml. of distilled water and thereafterwith a solution of CaCl (0.1 g.) in 100 ml. of distilled water, thefinal solution being sterilized by filtration, the final pl-l beingcomprised between 7.2 and 7.4), this latter being renewed several timesup to elimination of the nitrite anion. A sample is titrated and storedat C. The titration is performed by the tube end-point dilution methodin primary foetal bovine kidney tissue culture at the nonpermissivetemperature (39 C 1 1 C) using two tubes per dilution.

After a 2 week incubation period, the titer is recorded and the samplestored at 70 C is diluted to contain 1 TCID /0.2 ml. This diluted sampleis inoculated in 28 primary foetal bovine kidney tissue culture tubesusing 0.1 ml. inoculum per tube. The tubes are incubated at thepermissive temperature (30 C/i 1 C). After various incubation periodsranging from 7 to 17 days, 10 inoculated tubes show a typical bovineAdeno 3 virus cytopathogenic effect; these tubes are labelled 1 to 10and stored at 70 C. Parallel titrations of these 10 positive samples areperformed at the permissive temperature (30 C) and at the non-permissivetemperature (39 C). Samples exhibiting a significant difference in titerbetween the permissive and the non permissive temperatures are furthercloned twice by limit dilution passages.

The obtained virus is then multiplied by two passages in primary foetalbovine kidney (PFBK) cell cultures, as follows: Bovine foetuses are usedas kidney donors. The kidneys are removed under aseptic conditions.Minced kidney tissue is washed in phosphate buffer saline (consisting ofNaCl (8 g.); KCl (0.2 g.); Na H- PO (1.15 g.); KH PO (0.2 g.) indistilled water (up to 800 ml.) mixed with a solution of MgCl .6H O inml. of distilled water and thereafter with a solution of CaCl (0.1 g.)in 100 ml. of distilled water, the final solution being sterilized byfiltration, the final pH being comprised between 7.2 and 7.4) andtrypsinized with a buffered saline solution of trypsin (2.5 g./l.) andthe mixture is continuously stirred for 10 minutes at a temperature of37 C. The liquid is then poured off and replaced by an equal volume offresh trypsin solution. Trypsinization is then continued with stirringuntil exhaustion of the tissue, the cells suspended in the liquid beingremoved from time to time and then centrifuged at 1,000 rpm. anad thecell sediment is suspended in growth medium (Eagles basal mediumsupplemented with 10 virus screened calf serum, 100 units of sodiumpenicillin G and 100 mcg. of streptomycin sulfate 5 per ml.) to provideabout 200,000 cells per ml.

One ml. samples of the cell suspension are inoculated into sterile tubesand incubated for 4 to 5 days at 37 C. At the end of this initialincubation period, the growth medium is removed and replaced in eachtube by 1.5 ml. of Eagles basal medium containing only 2 agamma virusscreened calf serum. Ten tubes are inoculated with 0.2 ml. of thehereabove obtained virus suspension and incubated at 35 C for periodsvarying between 7 and 14 days. The virus growth is evidenced by typicalcytopathogenic effect and a passage of the virus to a second primaryfoetal bovine kidney (PFBK) tissue culture is carried out when about 50of the cells exhibit said cytopathogenic effect. The supernatant fluidis then harvested and a 0.2 ml. sample thereof is used as inoculum forthe next passage.

The supernatant fluids of the second passage are harvested, pooled anddiluted in a volume ratio of 1:2 with a stabilizing solution known as FGsolution and consisting of casitone 60 g.; sucrose 100 g.; sodium TABLEI Virus yield TCID (expressed Difference Strain in l gw/0.1 m1.) Between395 C at 35 C at 39.5 C and 35 C WBR 1/7 6.5 6.5 0

Eunice 4.5 1.7 2.8

The stability of the ts character of Eunice strain has been demonstratedin vitro as follows:

Eunice strain has been passaged in primary foetal bovine kidney (PFBK)cell cultures at a permissive temperature (from 30 to 35 C). The resultsare summarized in Table 11 showing that the ts character remained stablethroughout 7 passages at 30-35 C while the growth of the strain isconsiderably reduced at 39.5C.

TABLE II Passage Virus titer TCID,-,.. (in 1og /0.1 ml.)

level at permissive temperature at non-permissive (SO-35 C) temperature(395C) Moreover, the ts character of the virus produced at thenon-permissive temperature (395 C) :was also tested. As indicated inTable II, Eunice strain poorly growths at the non-permissive temperatureand the results of Table III indicate that the so-produced virus haskept its ts character.

TABLE III Titer (10g ,TClD,-,"/0.l ml. at permissive and non-permissivetemperatures) of virus initially produced at 395 C.

Bovine foetal kidneys are removed under aseptic conditions, minced andwashed in phosphate buffer saline (consisting of NaCl (8 g.); KC] (0.2g.); Na l-I- PO, (1.15 g.); Kl-l PO (0.2 g.) in distilled water (up to800 ml.) mixed with a solution of MgCl .6l-I O in 100 ml. of distilledwater and thereafter with a solution of CaCl (0.1 g.) in 100 ml. ofdistilled water, the final solution being sterilized by filtration, thefinal pH being comprised between 7.2 and 7.4) and trypsinized with abuffered saline solution of trypsin (2.5 g/l.) and the mixture iscontinuously stirred for 10 minutes at a temperature of 37 C. The liquidis then poured off and replaced by an equal volume of fresh trypsinsolution. Trypsinization is then continued with stirring untilexhaustion of the tissue, the cells suspended in the liquid beingremoved from time to time and then centrifuged at 1,000 rpm. for 5minutes and the cell sediment is suspended in growth medium (Hanks basicsalt solution supplemented with 10 virus screened calf serum. 0.5lactalbumin hydrolysate, 0.1 yeast extract and 50 mcg. of neomycinsulfate per ml.) to provide about 200,000 cells per ml.

Aliquots (one ml.) of the cell suspension are inoculated into 500 squarecentimeter culture flasks and incubated for 4 to 5 days at 37 C. At theend of this initial incubation period, the growth medium is removed andthe cell monolayer is washed twice with a maintenance medium consistingof Earles basic salt solution containing 0.5 lactalbumin hydrolysate;0.1 yeast extract; 0.1 tryptose phosphate broth and 50 mcg. of neomycinsulfate'per ml.

Each bovine foetal kidney cell culture flask is inoculated with one ml.of a suspension of bovine Adeno 3 virus Eunice strain in distilled waterand containing about 2.10 TCID /ml. (i.e. at the multiplicity index of0.1). Maintenance medium (same composition as the above washings) isadded to each flask and the culture is incubated at a temperature of 35C for a period of time sufficient to permit growth of a large amount ofvirus, i.e. for at least 5 to 10 days as evidenced by typicalcytopathogenic effect of bovine Adeno 3 virus.

The supernatant fluids are then harvested, pooled and diluted in avolume ratio of 1:2 with a stabilizing solution known as FG solution andconsisting of casitone 60 g.; sucrose 100 g.; sodium phosphate dibasic(M/ 15) ml.; potassium phosphate monobasic (M/l5) 25 ml.; monopotassiumglutamate 20 g.; distilled water sufficient to produce one liter.

The preparation is distributed into glass vials containing either 2.10TCID or multiples thereof and the vails are freeze-dried and sealed forconstituting either single or multiple doses. After reconstitution byadding 4 m1. of waterper dosis, the vaccine is administered as coarsespray to the animal.

a. Vaccination program.

The so-obtained vaccine was inoculated by intranasal way to nine animals(6 months old calves) at a dosage unit of 2.10 TCl m (two ml. pernostril). The animals were selected for their variable serological statereproducing conditions existing within the bovine population in Europe(for instance, CH. Ludwig and H. Liebermann Monatsch. Vet. Med. 25. 229,1970 and G.

in order to eliminate residual bovine serum. For each dilution. four orfive tubes were inoculated with 0.1 ml. The cultures were incubated at35 C- and the titers were determined by estimation of the cytopathogeniceffect. after a 14 day incubation period.

As indicated in Table V, testing of the ts character of the virusre-isolated from the nose of the vaccinated animals demonstratesstability of the ts character of the vaccine virus Eunice strain afterin vivo passage.

TABLE V ND not determined.

Wellemans et J. LeunenzVlaams Diergen. Tijdschr. 3, 125, 1968).

b. Virus re-isolation after vaccination.

Samples obtained by nasal swabbing were checked for presence of virus inthe nasal cavities. The virus re-isolated from the nose of thevaccinated animals has been passaged in culture tubes of secondaryfeotal bovine tissue incubated for 14 days at 35 C, all the negativesamples being subpassaged in the same conditions and culture system.

The final results are indicated in Table IV.

d. Virus re-isolation after challenge.

All the animals were challenged 27 days after vaccination with WBR l/7strain, using TCID per nostril.

The virus reisolated from the nose of the vaccinated animals has beenpassaged in culture tubes of secondary foetal bovine kidney tissueincubated for 14 days at 395 C, all the negative samples beingsubpassaged in the same conditions and culture system.

The final results are indicated in Table VI.

TABLE VI TABLE IV Reference Day of reisolation after vaccination numberof Animal status the animal 3 4 5 6 7 l() l l 70 indirect control 72indirect control 71 direct control 73 direct control 74 vaccinated 75vaccinated 76 vaccinated 77 vaccinated 78 vaccinated 79 vaccinated 80vaccinated 81 vaccinated 82 vaccinated m (+)=positive in subpassage.

These results indicate that the vaccine virus was reisolated from allthe vacinated animals, veal 75 excepted. Re-isolation was obtained from3 to 6 days post-vaccination. Among the re-isolations, four wereobtained by subpassages. As resulting from the virological examinationconducted during the 11 day period following vaccination, the directcontrols were not contaminated by the vaccinated animals. Tentatives ofre-isolation from the feces were all negative and demonstrate innocuityof the vaccine of the invention due to the restricted multiplication ofthe vaccine virus strain; i.e. in the upper respiratory tract.

0. Ts character of the vaccine virus re-isolated after vaccination.

Titration and determination of the ts character were performed insecondary foetal bovine kidney tissue cultures, said btllthres beingwashed before inoculation Reference Day of number of Animal statusreisolation after challenge the animal 10 7O indirect control 72indirect control 71 direct control 73 direct control 74 vaccinatedvaccinated 76 vaccinated 77 vaccinated 78 vaccinated 79 vaccinated 8Ovaccinated 8| vaccinated 82 vaccinated (+)=positi\'e in subpassage.

The results of Table VI indicate that the challenge. virus was onlyisolated from two among the nine vaccinated animals and only on thethird day after challenge. In contrast, virus was recovered from thenose of all control animals. Particularly, indirect controls (70 and 72)had three positive samples on a total of five, during the day postchallenge observation period.

Table VI demonstrates that there is no multiplication of the challengevirus in the nasal cavities of the vaccinated animals.

e. Seroconversion after vaccination.

The serological testing was performed by seroneutralization using theconstant virus 100 TCID J/serum dilution technique, the first dilutionbeing 1/4.

The sera were previously inactivated by heating at 56 C for minutes andeach serum dilution was brought into contact with the virus for one hourat C before being distributed into culture tubes (0.2 ml. per tube)using four tubes of each serum dilution. The inoculated tubes wereincubated at 35 C and serum titers were determined after a 14 dayincubation period.

The results of the seroneutralization test are given in Table VII.

TABLE VIlI Reference Neutralization index (days number of Animal statusversus vaccination) the animal 1 1 l 70 indirect control 1 0 l I 72indirect control 0 0 ND ND 71 direct control 3 0 0 0 73 direct control 00 0 0 74 vaccinated 0 3 3 3 75 vaccinated 1 4 4 4 76 vaccinated 0 2 ND 277 vaccinated 0 3 3 (1 78 vaccinated 0 1 1 2 79 vaccinated 0 3 3 1 80vaccinated 0 3 l 2 81 vaccinated 0. 3 ND 3 82 vaccinated 0 1 1 0 ND notdetermined.

Table VIII indicates that 11 days after vaccination with a vaccine ofthis invention, six animals of nine show an important antibody level(corresponding to neutralization indexes of 3 or more), while theneutralization indexes of the three other vaccinated animals are l or 2.These antibodies persist for the whole 27 TABLE VII Reference Day ofsampling versus vacci number of Animal status nation day the animal 1 1327(chal 42 l 15 days lenge) alter chall.

70 indirect control 4 8 4 16 72 indirect control 64 64 64 256 71 directcontrol 128 128 256 512 73 direct control 128 128 128 1024 74 vaccinated32 -3256 512 512 75 vaccinated 128 3256 31024 $2048 76 vaccinated 32S256 512 1024 77 vaccinated 3 128 $256 256 256 78 vaccinated 32 $256 256256 79 vaccinated 32 $256 1024 2048 80 vaccinated 16 $256 512 5 I 2 81vaccinated 9128 3256 256 048 82 vaccinated 8 128 128 128 These resultsshow that animals having an antibody titer of 128 or less at the time ofvaccination already reacted 13 days after vaccination. In contrast, thedirect or indirect controls (except animal 71) does not show anysignificant increase of tite r during the same observation period.

The results after challenge indicate a significative increase ofantibodies titer among all the control animals (animal 71 excepted)while no significative increase was observed among all the vaccinatedanimals (animal 81 excepted).

The results demonstrate the high antigenicity of the vaccine accordingto the invention.

f. Serological characteristics of nasal secretions.

day observation period, except for animals 77 and 82.

No local antibodies were found in the direct control animals no. 71 and73.

Antibodies were detected before vaccination in three animals of whichone was vaccinated. Only the vaccinated animal (no. showed a significantincrease of the neutralization index 1 1 days after vaccination.

The results in conjunction with those of Table VI demonstrate the valueimmunogenicity of the vaccine according to the invention.

g. Specificity of the local antibodies induced by vaccination.

The specificity of the local antibodies induced by vaccination wasdetermined on concentrated pools of the nasal samplings on the llth,17th and 27th days after vaccination. Neutralization tests of thesepools were performed against bovine Adeno 1 virus and bovine Adeno 3virus.

TABLE IX Reference Neutralization index of the number of Animal statuspools after vaccination. the animal Bovine Bovine Adeno 3 Adeno 1 71direct control 0 0 73 direct control 0 0 74 vaccinated 2 0 75 vaccinated4 0 77 vaccinated 3 0 79 vaccinated 3 0 TABLE lX-continued ReferenceNeutralization index of the number of Animal status pools aftervaccination. the animal Bovine Bovine Adeno 3 Adeno l 80 vaccinated 2 81vaccinated 3 82 vaccinated l W The results are indicated in thefollowing Table X.

TABLE X Reference Serological response expressed number of the animalsAs seroneutralizing antibodies As haemagglutination titer for inhibitiontiter for Adeno 3 virus [BR virus Pl virus before after before afterbefore after vaccivacci vaccivaccivaccivaccination nation nation nationnation nation l 4 16 O 2 8 I28 2 l6 32 O 2 8 32 3 4 32 O 8 8 l28 4 8 64O l 32 32 As appearing from Table IX, the antibodies detected in thenasal secretions are specific to bovine Adeno 3 25 The additional bovineFl and [ER viruses did not virus. There is no cross-reaction with bovineAdeno 1 virus.

h. Symptomatology.

Starting from the vaccination date, all the animals were observed dailyfor temperature, clinical examination for eventual ocular, respiratoryand digestive symptoms. All the observations were normal and thisabsence of any clinical symptom, notwithstanding the activemultiplication of the vaccine strain, demonstrates the innocuity of thevaccine according to the invention.

EXAMPLE 3 The technique is that described in example 2 but the harvestedsupernatant fluids diluted with the said stabilizing solution aresupplemented with the RLB 103 temperature-sensitive mutant strain ofbovine parainfluenza 3 (P13) virus described by N. Zygraich, M. Lobmannand C. Huygelen in J. Hyg. Camb. 70, 229-34, 1972 plus the RLB 106temperature-sensitive mutant strain of infectious bovine rhinotracheitis(IBR) virus (Research in Veterinary Science, in press) and the mixtureis distributed into glass vials containing 10" TCID of bovine Adeno 3virus Eunice strain, TCID of bovine PI3 virus and 10 TCID of lBR virusor multiples of each. The vials are freeze-dried and sealed forconstituting either single or multiple doses. After reconstruction byadding 4 to 5 ml. of

interfere with the Adeno 3 vaccination take and, inversely, the Adeno 3virus did not interfere with seroconversion of the animals of the bovineP1 and [BR viruses.

We claim:

1. A live virus bovine adenovirus intranasal vaccine protective againstbovine adenovirus type 3 infection comprising an effective intranasaldose of a temperature-sensitive nitrous acid mutant of the pathogenicbovine adenovirus, said mutant having a cut-off temperature of fromabout 39 to 40C and being able to multiply in the upper respiratorytract of a bovine animal but having inhibited replication in the lowerrespiratory tract and internal organs of the body, and a pharmaceuticalintranasal diluent.

2. A vaccine according to claim 1, in which the virus is a mutant ofbovine adenovirus type 3.

3. A vaccine according to claim 2, in which the dose is at least 2 X 10TCID 4. A vaccine according to claim 2, in freeze-dried form.

5. A process of immunizing a suscetible cow against infection withbovine adenovirus type 3 comprising intranasally administering to saidcow an effective dose of a vaccine according to claim 1.

6. A process of immunizing a susceptible cow against infection withbovine adenovirus type 3 comprising intranasally administering to saidcow an effective dose of a vaccine according to claim 2.

1. A LIVE VIRUS BOVINE ADENOVIRUS INTRANASAL VACCINE PROTECTIVE AGAINSTBOVINE ADENOVIRUS TYPE 3 INFECTION COMPRISING AN EFFECTIVE INTRANASALDOSE OF A TEMPERATURE-SENSITIVE NITROUS ACID MUTANT OF THE PATHOGENICBOVINE ADNOVIRUS, SAID MUTANT HAVING A CUT-OFF TEMPERATURE OF FROM ABOUT39* TO 40*C AND BEING ABLE TO MULTIPLY IN THE UPPER RESPIRATORY TRACT OFA BOVINE ANIMAL BUT HAVING INHIBITED REPLICATION IN THE LOWERRESPIRATORY TRACT AND INTERNAL ORGANS OF THE BODY, AND A PHARMACEUTICALINTRANASAL DILUENT.
 2. A vaccine according to claim 1, in which thevirus is a mutant of bovine adenovirus type
 3. 3. A vaccine according toclaim 2, in which the dose is at least 2 X 106 TCID50.
 4. A vaccineaccording to claim 2, in freeze-dried form.
 5. A process of immunizing asuscetible cow against infection with bovine adenovirus type 3comprising intranasally administering to said cow an effective dose of avaccine according to claim
 1. 6. A process of immunizing a susceptiblecow against infection with bovine adenovirus type 3 comprisingintranasally administering to said cow an effective dose of a vaccineaccording to claim 2.